PROJECT SUMMARY Background: Class switch recombination and somatic hypermutation are two B lymphocyte specific processes that mediate antibody diversification. Activation Induced Cytidine Deaminase (AID) is essential for initiating both of these processes by deaminating cytidine residues in immunoglobulin (Ig) loci DNA. Despite its specific and indispensible function in the Ig loci, AID has been demonstrated to also deaminate non-Ig locus genes, catalyzing various oncogenic translocations that manifest in tumorogenesis. Recent work has indicated that AID's DNA deamination activity requires its association with transcriptionally stalled RNA polymerase II and the RNA exosome complex. RNA exosome is a cellular non-coding RNA processing and/or degradation macromolecular complex. It is postulated that RNA Exosome's activity is mediated by specific cofactors and sequence characteristics of the target RNA. How RNA exosome's RNA processing activity facilitates AID mediated DNA deamination is a question we seek to address in this application. Objective/Hypothesis: In his proposal, we will determine how RNA exosome activity on transcripts generated in the IgH locus and the rest of the B cell genome generates single-stranded DNA structures following stalling of RNA polII complex and depletion of nucleosomes. Single-strand DNA structures are suitable AID substrates. Specific aims: Aim 1: Are the DIS3 and Exosc10 RNase subunits of RNA exosome complex important for AID activity?; AIM 2: How does RNA exosome substrate antisense RNA (xTSS-RNA and asRNA) promote AID targeting in the B cell genome? AIM 3: To evaluate the role of RNA exosome cofactor Mtr4 (and Senataxin) in the mutagenesis of both strands of DNA in the IgH locus and other regions of the B cell genome. Study Design: Using mouse models that are deficient in RNA exosome RNA degradation activity, we will identify regions in germinal center derived B cell genome that express RNA exosome substrate non-coding RNAs and are also mutated by AID. We will evaluate the mechanism of formation of single-strand DNA structures following localized chromatin remodeling at these identified AID target DNA sequences. We have also generated a mouse model in which the RNA exosome subunit, Exosc3, has been tandem-tagged for high affinity RNA exosome complex purification. Using B cells from these mice, we have purifed RNA exosome complex co-factors and identify them by LC-MS/MS. We will evaluate the role of RNA exosome co-factor(s) in stimulating AID/RNA exosome complex function on both strands of transcribed DNA substrate. Disease Relevance: AID initiates various malignancies in B cells due to its aberrant DNA mutagenesis activity. Proposed studies leads to a better understanding of the mechanisms initiating AID dependent oncogenesis in B lymphocytes (specially in context of DLBCL and Multiple Myeloma), as well as, have direct implications in understanding B lymphocyte based immunodeficiency syndromes like Hyper-IgM syndrome 2.